An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration

Abstract

Adult stem cells sustain tissue maintenance and regeneration throughout the lifetime of an animal1,2. These cells often reside in specific signalling niches that orchestrate the stem cell’s balancing act between quiescence and cell-cycle re-entry based on the demand for tissue regeneration2–4. How stem cells maintain their capacity to replenish themselves after tissue regeneration is poorly understood. Here we use RNA-interference-based loss-of-function screening as a powerful approach to uncover transcriptional regulators that govern the self-renewal capacity and regenerative potential of stem cells. Hair follicle stem cells provide an ideal model. These cells have been purified and characterized from their native niche in vivo and, in contrast to their rapidly dividing progeny, they can be maintained and passaged long-term in vitro5–7. Focusing on the nuclear proteins and/or transcription factors that are enriched in stem cells compared with their progeny5,6, we screened ~2,000 short hairpin RNAs for their effect on long-term, but not short-term, stem cell self-renewal in vitro. To address the physiological relevance of our findings, we selected one candidate that was uncovered in the screen: TBX1. This transcription factor is expressed in many tissues but has not been studied in the context of stem cell biology. By conditionally ablating Tbx1 in vivo, we showed that during homeostasis, tissue regeneration occurs normally but is markedly delayed. We then devised an in vivo assay for stem cell replenishment and found that when challenged with repetitive rounds of regeneration, the Tbx1-deficient stem cell niche becomes progressively depleted. Addressing the mechanism of TBX1 action, we discovered that TBX1 acts as an intrinsic rheostat of BMP signalling: it is a gatekeeper that governs the transition between stem cell quiescence and proliferation in hair follicles. Our results validate the RNA interference screen and underscore its power in unearthing new molecules that govern stem cell self-renewal and tissue-regenerative potential.

Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration

“…Gene hits were scored by criteria similar to those used in other recent work (3,20), according to whether there was a progressive change of 1.7-fold of normalized shRNA read abundance for at least two shRNAs targeting the given gene, with no shRNA showing a contradictory change, or whether shRNAs disappeared entirely from all cell phenotypes after differentiation (Table S2). The 1.7-fold on the log 2 plot represents almost two SDs, so that a point above 1.7-fold the mean value has less than between a 5% and 6% chance of being a result of chance.…”

p53 suppression partially rescues the mutant phenotype in mouse models of DiGeorge syndrome

“…Tbx1 mutation is associated with reduced cell proliferation in several other tissues, for example the pharyngeal endoderm and the otic epithelium (7,8). In addition, Tbx1 has also been implicated in the expansion of dental stem cells (9) and self-renewal of hair follicle stem cells (10). Based on this knowledge, we sought genetic strategies that could counteract the effects of reduced dosage of Tbx1.…”

Tbx3 represses bmp4 expression and with Pax6 is required and sufficient for retina formation

“…Xenopus Tbx1-Tbx5 are all expressed during neural development and have non-identical expression patterns (Li et al, 1997;Takabatake et al, 2000;Sasagawa et al, 2002;Showell et al, 2006). Tbx1 suppresses BMP signaling in the mouse hair follicle stem cell niche and cardiomyocytes, while Tbx4 and Tbx5 both repress BMP2 during chick limb development (Rodriguez-Esteban et al, 1999;Fulcoli et al, 2009;Chen et al, 2012). Tbx2, in contrast, activates BMP signaling in the mouse limb bud (Farin et al, 2013).…”